N,N,N-Tris(tert-butoxycarbonyl)-l-arginine: five isoforms whose obtainment depends on procedure and scrupulous NMR confirmation of their structures

Article abstract of DOI:10.1007/s11164-017-3199-6

l-arginine is often covalently linked to vectors for gene or drug delivery as a means of increasing their transfection activity and reducing toxicity. This strategy relies on the protection of basic nitrogen atoms, for example, by employing the tert-butoxycarbonyl group. Our aim in the present work was to prepare the widely described ^αN,^ωN,^ω′N-tris(tert-butyloxycarbonyl)-l-arginine as a single isomer in high yield and with high levels of purity for use in the esterification of dendrimers with several peripheral hydroxyl groups. Following three reported protocols which assured this goal, we observed the unexpected formation of four additional isomers. Using the first procedure, ^αN,^ωN,^ω′N-tris(tert-butyloxycarbonyl)-l-arginine was never obtained. The second procedure delivered the desired compound as a mixture of geometric isomers (E/Z), while the third protocol led to a single isomer in high yield and purity, but with an unreported symmetrical structure. Since Boc protection is transient, this discovery would seem to be of little interest, but preliminary results from an ongoing investigation of the behavior of each of the isomers obtained in the esterification reactions of interest has shown that their reactivity depends on their structure. Although this research is ongoing, here we report a detailed description of these unexpected results, along with an NMR investigation focusing on the double-bond geometry and position which enabled confirmation of the structures.

Full text of DOI:10.1007/s11164-017-3199-6

Res Chem Intermed (2018) 44:1811–1832
https://doi.org/10.1007/s11164-017-3199-6
N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five
isoforms whose obtainment depends on procedure
and scrupulous NMR confirmation of their structures
Silvana Alfei1
Sara Castellaro1
•
Received: 9 May 2017 / Accepted: 11 November 2017 / Published online: 28 November 2017
Ó Springer Science+Business Media B.V., part of Springer Nature 2017
Abstract L-arginine is often covalently linked to vectors for gene or drug delivery
as a means of increasing their transfection activity and reducing toxicity. This
strategy relies on the protection of basic nitrogen atoms, for example, by employing
the tert-butoxycarbonyl group. Our aim in the present work was to prepare the
0
a
widely described N,xN,x N-tris(tert-butyloxycarbonyl)-L-arginine as a single iso-
mer in high yield and with high levels of purity for use in the esterification of
dendrimers with several peripheral hydroxyl groups. Following three reported
protocols which assured this goal, we observed the 0unexpected formation of four
additional isomers. Using the first procedure, aN,xN,x N-tris(tert-butyloxycarbonyl)-
L-arginine was never obtained. The second procedure delivered the desired com-
pound as a mixture of geometric isomers (E/Z), while the third protocol led to a
single isomer in high yield and purity, but with an unreported symmetrical structure.
Since Boc protection is transient, this discovery would seem to be of little interest,
but preliminary results from an ongoing investigation of the behavior of each of the
isomers obtained in the esterification reactions of interest has shown that their
reactivity depends on their structure. Although this research is ongoing, here we
report a detailed description of these unexpected results, along with an NMR
investigation focusing on the double-bond geometry and position which enabled
confirmation of the structures.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11164-
017-3199-6) contains supplementary material, which is available to authorized users.
&
Silvana Alfei
alfei@difar.unige.it
1
Dipartimento di Farmacia, Sezione di Chimica e Tecnologie Farmaceutiche e Alimentari,
`
Universita degli Studi di Genova, Viale Cembrano 4, 16148 Genoa, Italy
123

1812
S. Alfei, S. Castellaro
Keywords L-arginine Á Boc protecting group Á Unreported and unexpected
results Á NMR characterization Á Rotamers Á Tautomers Á Regioisomers
Introduction
L-arginine is a semi-essential amino acid of great interest in several areas of
research. For instance, manipulation of L-arginine metabolism may be used as a
treatment strategy in chronic kidney disease (CKD) and associated disorders,
including renal ablation, ureteral obstruction, puromycin-induced nephrosis and
nephropathy secondary to diabetes, and complications associated with salt-sensitive
hypertension, radiocontrast agents, and aging [1, 2]. In addition, L-arginine has
garnered growing interest for use in nanomedicine, gene therapy and drug delivery.
Together with L-lysine, the application of L-arginine for the functionalization of
synthetic vectors is particularly appealing for several reasons, and L-arginine is
especially attractive in this regard. Peptides that are rich in L-arginine are widely
used as carriers for genetic material [3] and siRNA [4], and there are many
examples of dendrimers exhibiting improved cellular uptake of siRNA [5, 6] and
more efficient transfection activity due to the presence of L-arginine moieties [7, 8].
Hyperbranched copolymers that are L-arginine-rich (P-lys-arg0.20) have demon-
strated significantly enhanced transfection efficiency, which is dependent on the
arginine content of the polymer itself, under all conditions studied, and lower
toxicity in COS-7 cells [9]. Our research is aimed at preparing non-cytotoxic
polyester dendrimers decorated with amino acids [10, 11] in order to obtain
polycationic macromolecules capable of DNA or RNA binding and cell transfection
for the purpose of gene therapy. Thus, it became necessary to functionalize the
intermediate dendrons with L-arginine. To mitigate complications during covalent
coupling, the basic nitrogens of L-arginine must be protected. The NO2-protecting
0
group of commercially available aN-tert-butoxycarbonyl-x N-NO2-L-arginine would
be sensitive to the reductive conditions used during the generational growth of our
dendrimers. Therefore, tert-butyloxycarbonyl (Boc2O), which can be readily
eliminated in the presence of anhydrous acid, emerged as the most suitable protect-
ing group. Although the selective preparation of tert-butyloxycarbonyl-L-arginine
derivatives protected only at the necessary basic centers (a and x0) has not been
previously documented, the literature contains exhaustive reports regarding
0
aN,xN,x N-tris(tert-butyloxycarbonyl)-L-arginine [12–16]. We needed to access
the tert-butyloxycarbonyl-L-arginine derivative in high yield and in isomerically
pure form to avoid the obtainment of complex mixtures with convoluted NMR
spectra upon linkage to dendrimers. To this end, we performed three previously
reported procedures [12, 15, 16] that assured this goal. However, the results we
observed were much more complex than anticipated, and four additional isomers
were obtained, a finding we believe worth communicating. In this paper, therefore,
we report our findings, which contrast with those described in the literature: instead
of yielding the same product, each of the three protocols led to different isomers.
This discovery from the initial results of ongoing research revealing the interplay
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1813
between structure and reactivity will enable researchers to select a procedure that
best suits their objective. We have provided a complete spectral characterization of
each isomer obtained, highlighting the primary spectral differences that allowed
assignment of the structures, thereby providing a powerful tool for recognizing and
distinguishing the isomeric forms. Lastly, we provide a detailed description of
various unreported complications related to the applied procedures.
Experimental
General
Melting points, determined on a Leica Galen III hot-stage apparatus, are
uncorrected. FTIR spectra were recorded as film or KBr pellets on a PerkinElmer
Spectrum System 2000 spectrophotometer. 1H and 13C NMR spectra were acquired
on a Bruker Avance DPX 300 spectrometer at 300 and 75.5 MHz, respectively,
using CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS) as internal
reference. 13C NMR signals were assigned through DEPT-135 experiments.
Coupling constants are given in hertz. Fully decoupled 13C NMR spectra are
reported. Chemical shifts are reported in d (parts per million) units relative to the
internal standard TMS (d = 0.00 ppm), and the splitting patterns are described as
follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and brs (broad
signal). Thin-layer chromatography (TLC) for routine monitoring of the course of
reactions and confirming the purity of analytical samples was conducted on
aluminium-backed silica gel plates (Merck DC silica gel 60 F254), and spots were
detected under UV light and/or by ninhydrin solution (0.2% in ethanol) heated on a
stove at 100 °C. Preparative thin-layer chromatography (PLC) was performed on
0.5-mm, 20 9 20 cm Merck silica gel plates and was visualized by UV light. Merck
silica gel (0.040–0.063 mm) was used. Elemental analysis was performed using an
EA 1110 Elemental Analyser (Fison Instruments, Milan), with values obtained
within
0.4% of the theoretical values. Organic solutions were dried over
anhydrous sodium sulphate and were evaporated using a rotatory evaporator
operating at a reduced pressure of roughly 10–20 Torr. All chemicals were
purchased from Sigma-Aldrich or Merck and used without further purification.
The solvents were dried and distilled according to standard procedures.
Petroleum ether refers to the fraction with a boiling point of 40–60 °C.
aN-tert-butoxycarbonyl-L-arginine (2) [17, 19], aN-tert-butoxycarbonyl-L-or-
nithine (4) [20, 21], N,N0-bis(tert-butoxycarbonyl)guanidine (8) [22] and N,N0-
bis(tert-butoxycarbonyl)triflyl guanidine (9) [22] were prepared according to a
known procedure, and the experimental and analytical data are available in Online
Resource (Experimental procedure S8, S9, S10, S11).
123

1814
S. Alfei, S. Castellaro
0
a
a
Synthesis of N,dN,xN-tris(tert-butoxycarbonyl)-L-arginine (3a), N,dN,x N-
tris(tert-butoxycarbonyl)-L-arginine (3b) and N,xN-bis(tert-
butoxycarbonyl)-L-arginine (3c) [23, 24]
a
0
a
Following a reported protocol for the preparation of N,xN,x N-tris(tert-butoxycar-
bonyl)-L-arginine [12], 2 (1.3 g, 4.5 mmol), 2 N NaOH (27 mL), dioxane (20 mL)
and di-tert-butyl-carbonate (Boc2O) (2.10 g, 9.7 mmol) were introduced into a
100-mL one-neck flask equipped with a magnetic stirrer. The solution was stirred
for 3 h and then cooled to 0 °C, and Boc2O was again added (1.10 g, 4.8 mmol),
along with dioxane (12 mL). The solution was stirred for 39 h at room temperature,
leading to the formation of a white suspension. The pH was brought to 7 by the
addition of 3 N HCl, which gave rise to a clear solution that was extracted with
ethyl acetate (AcOEt) (3 9 15 mL). The organic phase was dried over anhydrous
Na2SO4. After removal of the solvent under reduced pressure, a complex crude
product was obtained as determined by TLC (CHCl3/MeOH = 9/1), and the
composition of this crude material was first analyzed by 1H NMR. The crude
product was a mixture of at least two compounds, and so it was subjected to silica
gel column chromatography (h = 50 cm, ø = 3.5 cm) using a mixture of CHCl3/
MeOH = 9/1 as eluent for the fractions of interest. A mixture of aN,dN,xN-tris(tert-
0
a
butoxycarbonyl)-L-arginine (3a) and N,dN,x N-tris(tert-butoxycarbonyl)-L-arginine
0
(3b) eluted first, followed by aN,x N-bis(tert-butoxycarbonyl)-L-arginine (3c). After
complete characterization (IR, NMR, m.p. and elemental analysis), a sample of the
mixture 3a ? 3b (136.7 mg, 0.29 mmol) was again subjected to PLC using CHCl3/
MeOH = 9/1 as eluent, which afforded the major component 3a in high purity
(35.6 mg, 0.075 mmol, 26% PLC yield).
a
0
a
Mixture of N,dN,xN-tris(tert-butoxycarbonyl)-L-arginine (3a) ? N,dN,x N-tris
(tert-butoxycarbonyl)-L-arginine (3b) 412.4 mg, white solid (mixture 3a ? 3b in
1
ratio of 2.5:1 estimated by H NMR), Yield: 19%, m.p. 124–125 °C; FTIR (film)
3500–2400 (OH), 3421 (NH), 3332 (NH), 1720 (C=O acid ? C=O carbamate),
1
1639 (NH),1619 (NH) cm-1; H NMR (300 MHz, CDCl3): d 1.43 (s, 2.6H, CH3
Boc of 3b), 1.44 (s, 6.4H, CH3 Boc of 3a), 1.49 (s, 9H, CH3 Boc of 3a ? 3b), 1.51
(s, 2.6H, CH3 Boc of 3b), 1.52 (s, 6.4H, CH3 Boc of 3a), 1.58–2.00 (m, 4H,
bCHc2CH2), 3.87 (m, 2H, dCH2), 4.16 (m, 0.3H, aCHNH of 3b), (m, 0.7H, aCHNH of
a
3a), 5.69 (two intersected d, 1H, NH of 3a ? 3b), 8.20, 8.50 and 9.50 (s and brs,
0
x
2H, xNH ? x NH of 3a ? NH2 of 3b); 13C NMR (75.5 MHz, CDCl3): d 23.86 and
24.63 (CH2), 27.94 and 28.00, 28.21, 28.39 (CH3 Boc), 28.94 (CH2), 44.28
(dCH2NBoc), 53.26 and0 53.65 (aCHNHBoc), 79.32, 79.63, 79.70, 79.82, 84.09,
x
84.18 (C Boc), 153.83 x NC=O of 3b, 154.84 (aNC=O of 3a ? 3b and NC=O of
3a), 155.63 (dNC=O of 3b), 155.91 (dNC=O of 3a), 160.21 (C=NH of 3a), 162.65
(C=NBoc of 3b), 175.60 (C=O acid of 3a), 177.23 (C=O acid of 3b); Anal. Calc. for
C21H38N4O8 (474.55): C, 53.15%; H, 8.07%; N, 11.81%; found: C, 53.21%; H,
8.45%; N, 11.67%.
aN,dN,xN-Tris(tert-butoxycarbonyl)-L-arginine (3a) 107.4 mg, yellowish low-
melting glassy solid. Yield: 5%; FTIR (film) 3500-2400 (OH), 3421 (NH), 3332
(NH), 1720 (C=O acid ? C=O carbamate), 1639 (NH), 1619 (NH) cm-1; 1H NMR
(300 MHz, CDCl3): d 1.44 (s, 9H, CH3 Boc), 1.49 (s, 9H, CH3 Boc), 1.52 (s, 9H,
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1815
b
d
a
CH3 Boc), 1.58–1.87 (m, 4H, CH2cCH2), 3.87 (m, 2H, CH2), 4.30 (m, 1H, CH),
0
5.68 (d, J = 7.3 Hz, 1H, aNH), xNH and x NH not detected; 13C NMR (75.5 MHz,
CDCl3): d 24.64, 28.01, 28.21, 28.38, 28.89, 44.27, 53.58, 79.66, 79.88, 84.21,
154.84 (aNC=O ? NC=O carbamate), 155.89 (dNC=O carbamate), 160.20
(C=NH), 175.69; Anal. Calc. for C21H38N4O8 (474.55): C, 53.15%; H, 8.07%; N,
x
11.81%; found: C, 53.12%; H, 8.21%; N, 11.67%.
aN,xN,x N-Tris(tert-butoxycarbonyl)-L-arginine (3b) (indirectly derived spectral
data) 1H NMR (300 MHz, CDCl3): d 1.43 (s, 9H, CH3 Boc), 1.49 (s, 9H, CH3 Boc),
0
b
d
1.51(s, 9H, CH3 Boc), 1.58–2.00 (m, 4H, CHc2CH2), 3.87 (m, 2H, CH2), 4.16 (m,
1H, aCH), 5.71 (d, 1H, aNH), 8.50 and 9.50 (s and brs, 2H,xNH2 of 3b); 13C NMR
(75.5 MHz, CDCl3): d 23.86 (CH2) 27.94, 28.21, 28.39 (CH3 Boc), 28.94 (CH2),
44.28 (dCH2NBoc), 53.26 (aCHNHBoc), 79.32, 79.70, 84.09, (C Boc), 153.83,
154.84, 155.63, C=O carbamate, 162.65 (C=NBoc), 177.23 (C=O acid).
aN,xN-Bis(tert-butoxycarbonyl)-L-arginine (3c) [23, 24] 185.4 mg, white solid.
Yield 10%; m.p. 139 °C; FTIR (KBr) 3500–2400 (OH), 3421 (NH), 3332 (NH),
1720 (C=O acid ? C=O carbamate), 1639 (NH),1619 (NH) cm-1 1H NMR
;
(300 MHz, CDCl3): d 1.43 (s, 9H, CH3 Boc), 1.52 (s, 9H, CH3 Boc), 1.60–1.95 (m,
4H, bCHc2CH2), 3.29 (m, 1H, dCH2), 3.68 (m, 1H, dCH2), 4.10 (m, 1H, aCH), 5.60 (d,
a
d
J = 6.1 Hz, 1H, NH), 8.42 and 9.00 (s and brs, 3H, NH, xNH and x’NH); 13C
NMR (75.5 MHz, CDCl3): d 25.21, 27.96, 28.46, 30.08, 40.18, 53.54, 79.03, 83.99,
153.96 (C=O carbamate), 154.96 (C=O carbamate), 155.36 (C=NH), 177.68 (C=O
acid); Anal Calc. for C16H30N4O6 (374.22): C, 51.31%; H, 8.08%; N, 14.96%;
found: C, 51.23%; H, 8.28%; N, 15.27%.
0
a
Synthesis of N,xN,x N-tris(tert-butoxycarbonyl)-L-arginine (6)
0
a
Following a reported protocol for the preparation of N,xN,x N-tris(tert-butoxycar-
bonyl)-L-arginine [15], 4 (961.2 mg, 5.17 mmol), NaHCO3 (434.5 mg, 5.17 mmol),
H2O/dioxane 1/1 (23 mL) and S-methyl-N,N-bis(tert-butoxycarbonyl)-isothiourea
(5) (1.2 g, 4.1 mmol) were introduced into a 50-mL one-neck flask, and the reaction
mixture was stirred at 40 °C for 48 h. The reaction was monitored by TLC (CHCl3/
MeOH 9/1). Upon completion, the solvent was removed under reduced pressure,
yielding a solid which was dissolved in AcOEt (400 mL). The solution was washed
with 0.3 N HCl (26 mL) and H2O (6 9 20 mL), and the organic phase was dried
over anhydrous Na2SO4. The removal of the solvent under reduced pressure
provided the crude product, which was partially crystallized from diisopropyl ether
(DIPE)/petroleum ether 40–60 °C, while the non-crystallized material was
subjected to column chromatography to remove residual 5, affording 6 as a mixture
1
E/Z 2.2/1 as estimated by H NMR analysis. Following complete characterization
(IR, NMR, m.p. and elemental analysis), a sample of the mixture 6 E, Z (150.0 mg,
0.32 mmol) was again subjected to PLC using CHCl3/MeOH = 9/1 an eluent,
delivering the major component 6 E (110.10 mg, 0.23 mmol) and the minor product
6 Z (38.0 mg, 0.08 mmol, 97% PLC yield).
0
aN,xN,x N-Tris(tert-butoxycarbonyl)-L-arginine (6) 932.9 mg, white solid (mix-
ture of two rotamers E/Z 2.2/1 estimated by 1H NMR). Yield: 39%; m.p.
102–104 °C (DIPE/petroleum ether 40–60 °C), lit. 102–103 °C [15]; FTIR (KBr)
123

1816
S. Alfei, S. Castellaro
3500–2400 (OH), 3331 (NH), 1720 (C=O acid ? C=O carbamate), 1643 (NH),
1620 (NH) cm-1;1H NMR (300 MHz, CDCl3): d 1.43 (s, 2.8H, CH3 Boc of 6 Z),
1.44 (s, 6.2H, CH3 Boc of 6 E), 1.48 (s, 6.2H, CH3 Boc of 6 E), 1.49 (s, 6.2H, CH3
Boc of 6 E), 1.51 (s, 2.8H, CH3 Boc of 6 Z), 1.52 (s, 2.8H, CH3 Boc of 6 Z),
1.68–1.92 (m, 4H, bCH2cCH2), 3.41 (m, 2H, dCH2), 4.20 (m, 0.3H, aCH of 6 Z), 4.29
a
a
(m, 0.7H, CH 6 E), 5.37 (d, J = 7.7 Hz, 0.3H, NH of 6 E), 5.70 (d, J = 7.3 Hz,
0.7H, NH of 6 Z), 8.42 (brs,1H, NH),xNH not detected; 13C NMR (75.5 MHz,
CDCl3): d 25.27, 27.95, 28.07, 28.24, 28.36, 28.44, 29.84, 40.48, 53.36, 79.61 (C
Boc), 83.31 (two C 0 Boc overlapped), 153.23 (C=O carbamate), 155.79 (C=O
carbamate), 156.32 (x NC=O carbamate), 163.16 (C=NBoc), 175.41; Anal Calc. for
C21H38N4O8 (474.55): C, 53.15%; H, 8.07%; N, 11.81%; found: C, 53.27%; H,
8.47%; N, 011.77%.
a
d
aN,xN,x N-Tris(tert-butoxycarbonyl)-L-arginine (6 E) 684.7 mg, white solid.
Yield: 28%; m.p. 102 °C (DIPE/petroleum ether 40–60 °C); FTIR (KBr)
3500–2400 (OH), 3332 (NH), 1722 (C=O acid ? C=O carbamate), 1644 (NH),
1620 (NH) cm-1; H NMR (300 MHz, CDCl3): d 1.44 (s, 9H, CH3 Boc), 1.48 (s,
1
b
9H, CH3 Boc), 1.49 (s, 9H, CH3 Boc), 1.68–1.92 (m, 4H, CHc2CH2), 3.41 (m, 2H,
dCH2), 4.29 (m, 1H, CH), 5.37 (d, J = 7.7 Hz, 1H, NH), 8.42 (brs, 1H, NH),
a
a
d
xNH not detected; 13C NMR (75.5 MHz, CDCl3): d 25.27, 28.07, 28.24, 28.36,
29.84, 40.48, 53.36, 79.61 (C Boc), 83.31 (two C Boc overlapped), 153.23 (C=O
0
carbamate), 155.79 (C=O carbamate), 156.32 (x NC=O carbamate), 163.16
(C=NBoc), 175.41; Anal. Calc. for C21H38N4O8 (474.55): C, 53.15%; H, 8.07%;
N, 11.81%; found: C, 53.28%; H, 8.45%; N, 11.74%.
0
aN,xN,x N-Tris(tert-butoxycarbonyl)-L-arginine (6 Z) 236.3 mg, white solid.
Yield: 10% yield; m.p. 102–104 °C (DIPE/petroleum ether 40–60 °C); FTIR (KBr)
3500–2400 (OH), 3330 (NH), 1719 (C=O acid ? C=O carbamate), 1640 (NH),
1
1621 (NH) cm-1; H NMR (300 MHz, CDCl3): d 1.43 (s, 9H, CH3 Boc), 1.51 (s,
9H, CH3 Boc), 1.52 (s, 9H, CH3 Boc), 1.68–1.92 (m, 4H, CHc2CH2), 3.41 (m, 2H,
b
a
a
d
dCH2), 4.20 (m, 1H, CH), 5.70 (d, J = 7.3 Hz, 1H, NH), 8.42 (brs, 1H, NH),
xNH not detected; 13C NMR (75.5 MHz, CDCl3): d 25.27, 27.95, 28.24, 28.44,
29.84, 40.48, 53.36, 79.61 (C Boc), 83.31 (two C Boc overlapped), 153.23 (C=O
carbamate), 155.79 (C=O carbamate), 156.32 (x’NC=O carbamate), 163.16
(C=NBoc), 175.41; Anal. Calc. for C21H38N4O8 (474.55): C, 53.15%; H, 8.07%;
N, 11.81%; found: C, 53.25%; H, 8.41%; N, 11.79%.
0
a
Synthesis of symmetrical N,xN,x N-tris(tert-butoxycarbonyl)-L-arginine
(10)
0
a
Following a reported protocol for the preparation of N,xN,x N-tris(tert-butoxycar-
bonyl)-L-arginine [16], 4 (989.6 mg, 4.3 mmol), diisopropylethylamine (550.6 mg,
4.3 mmol), MeOH (40.2 mL) and 9 (1.64 g, 4.3 mmol) were introduced into a
100-mL four-neck flask. The reaction mixture was stirred for 7 days at room
temperature, after which the solvent was removed under reduced pressure, affording
a dark glassy solid, which was leached on a small silica column (h = 20 cm,
ø = 2 cm) using a mixture of CH2Cl2/MeOH = 4/1 as eluent. Removal of the
solvent under reduced pressure provided a spongy white solid, which was taken up
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1817
with AcOEt and hydrolyzed with 0.3 N HCl, washed with H2O and dried over
anhydrous Na2SO4. The material obtained after removal of the solvent was leached
again through a silica gel column (h = 15 cm, ø = 2 cm) using a mixture of
CH2Cl2/MeOH = 19/2 as eluent, which upon evaporation yielded 10.
0
aN,xN,x N-Tris(tert-butoxycarbonyl)-L-arginine (10) 2.04 g, white solid. Yield:
99%; m.p. 102–104 °C; FTIR (KBr) 3500–2400 (OH), 3422 (HN), 3332 (NH), 1720
(C=O acid ? C=O carbamate), 1639 (NH), 1619 (NH) cm-1; 1H NMR (300 MHz,
CDCl3): d 1.44 (s, 9H, CH3 Boc), 1.48 (s, 9H, CH3 Boc), 1.50 (s, 9H, CH3 Boc),
b
d
a
1.70–1.91 (m, 4H, CHc2CH2), 3.42 (brs, 2H, CH2), 4.26 (m, 1H, CH), 5.39 (br d,
0
1H, NH), 8.40–9.00 (br s, 2H, x NH ? NH); 13C NMR (75.5 MHz, CDCl3): d
25.18, 28.01, 28.11, 28.29, 29.57, 40.60, 53.25, 80.40, 80.65, 83.89, 153.09; 156.00;
156.08; 161.94; 175.68; Anal. Calc. for C21H38N4O8 (474.55): C, 53.15%; H,
8.07%; N, 11.81%; found: C, 53.30%; H, 8.27%; N, 12.00%.
a
x
Results
0
a
In an initial experiment we attempted to prepare the N,xN,x N-Tris(tert-butoxy-
carbonyl)-L-arginine in accordance with a procedure [12] that suggested two
a
different methods. We first chose the easiest to implement, which started from N-
tert-butoxycarbonyl-L-arginine (2), a compound that is easily prepared. The other
began with aN-tert-butoxycarbonyl-L-ornithine and used the commercially available
but very expensive 1H-pyrazole-N,N-bis(tert-butoxycarbonyl)-1-carboxamidine as
a
reagent. Then, L-arginine hydrochloride (1) was converted into N-tert-butoxycar-
bonyl-L-arginine (2) with Boc2O in 1 N NaOH/dioxane [17, 19], and 2 was obtained
as a white solid in 92% yield (Scheme 1), with spectral data in agreement with
literature data [18]. Next, 2 was treated with Boc2O in large excess again in 2 N
NaOH/dioxane [12] (Scheme 1), affording a rather complex crude in the form of a
1
white solid, which preliminary analysis by H NMR proved to be a mixture of two
0
Scheme 1 Literature [12] synthetic procedure for preparation of aN,xN,x N-tris(tert-butoxycarbonyl)-L-
arginine and the obtained products
123

1818
S. Alfei, S. Castellaro
or three compounds (Fig. S2 in Online Resource). This material was subjected to
column chromatography using chloroform/methanol (CHCl3/MeOH) 9/1 as eluent
to separate the unexpected components.
An accurate elution enabled separation of two main fractions. The head fraction,
1
a
after H and 13C NMR investigation, appeared to be a 2.5:1 mixture of N,dN,xN-
tris(tert-butoxycarbonyl)-L-arginine [19, 23] (we designated this compound 3a, and
it is commercially ava0ilable, but the NMR characterization is incomplete and
outdated) and aN,dN,x N-tris(tert-butoxycarbonyl)-L-arginine (namely 3b). The
mixture was obtained as a white solid in 19% yield. The ensuing fraction was
instead identified by NMR as N,xN-bis(tert-butoxycarbonyl)-L-arginine [23, 24]
a
(we designated this compound 3c, and only its predicted properties are available)
and was obtained as a white solid in 10% yield. From the mixture 3a ? 3b, we
managed to isolate the main component 3a in the form of a yellowish, low-melting,
glassy solid by PLC performed on 0.5 mm, 20 9 20 cm plates (CHCl3/MeOH 9/1).
1
In Fig. 1 the H NMR spectra of the mixture 3a ? 3b (top) and of the isolated
compound 3a (bottom) are shown; a figure including a significant portion of their
13C NMR spectra is available in the Online Resource (Fig. S3).
The structures of 3c and of 3a were assigned with certainty on the basis of the
chemical shift of the signal of the C=NH group (155.36 ppm for 3c and 160.21 ppm
for 3a). These data were in agreement with the data reported in the literature for
compounds containing the same functional group [25, 26]. Otherwise, the signal of
the C=NBoc group which was instead present in the structure of 3b should appear at
higher values of d [25].
Subtracting from the NMR spectra of the mixture 3a ? 3b the peaks belonging
to the isolated compound 3a, we managed to derive, even if indirectly, the signals of
3b, facilitating its characterization. In particular, between the signals deduced for
Fig. 1 A selected portion of the 1H NMR spectra of the mixture 3a ? 3b and of isolated 3a
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1819
the 13C NMR spectrum, a peak was detected at d = 162.65 ppm (C=NBoc group) in
accordance with the literature data [25]. In addition, the structures that we attributed
to 3a and 3b contained the dN-Boc group, whereas in the structure of 3c the dN was
not substituted. This was confirmed by the d of the signals, in both the proton
spectrum and carbon spectrum, corresponding to the dCH2 group of the compounds.
In the 1H NMR spectrum of the mixture 3a ? 3b, as in the spectrum of the isolated
3a, the signal was located at 3.9 ppm (Fig. 1), while in the spectrum of 3c it occurred at
3.50 ppm (Spectrum S19 in Online Resource). In the 13C NMR spectra the signals
were located at 44.47 ppm for 3a and 3b and at 40.18 ppm for 3c (Fig. S2 in Online
Resource). These data are all in agreement with the literature data [15, 16, 26] for
similar compounds: d [ 3.8 ppm in 1H NMR and [ 44 ppm in the 13C NMR
(dCHd2NBoc) [22] and d \ 3.5 ppm and around 40 ppm (dCH2NH) [15, 16, 26].
In addition, it is noteworthy that in the proton spectrum of 3c, the signal for the
dCHd2NH group (centered at 3.50 ppm) appeared as two very broad multiplets (see
spectrum S19 in Online Resource), since the diastereotopic protons in the d position
are coupled to each other as well as the protons in b position and the proton on the d
d
nitrogen atom. Otherwise, the signal for the CHd2NBoc group of 3a and 3b almost
appeared to be a broad triplet, as it was not affected by coupling with the proton of
the dNH group (Fig. 1). Finally in the 3c NMR spectra, the number of signals for the
CH3 and the quaternary carbons of the Boc group were obviously reduced to two
1
singlets that integrated to 18 protons in the H NMR spectrum, thus confirming the
presence of only two protecting groups (Fig. 2).
These outcomes were markedly different from the results reported in the
literature that we used as reference [12], which suggested that only a single product
0
a
identified as N,xN,x N-tris(tert-butoxycarbonyl)-L-arginine was obtained in 27%
yield after column chromatography. Although the reaction has been repeated several
times, this compound has never been obtained by us. The same misunderstanding
Fig. 2 Comparison between selected portions of 1H NMR and 13C NMR spectra of 3a and 3c
123

1820
S. Alfei, S. Castellaro
occurred in two recent papers [13, 14], which assured that under similar basic
0
conditions, aN,xN,x N-tris(tert-butoxycarbonyl)-L-arginine was obtained, without
the need for purification, in 64% yield. Similar results were reported in a paper from
1986 [23] and in another article from the same author in 1994 [19]. In our
laboratory, the structure of the major compound obtained [19, 23] was that of 3a,
and the reported 1H NMR data coincide with this structure, even if the melting point
(123–124 °C) and the physical state (crystalline form) of that compound agree with
those of the mixture of 3a ? 3b that we isolated with column chromatography.
Compound 3a, obtained as a pure isomer only after PLC (as assessed by 1H and 13
C
NMR spectra), was an oil which solidified over time, giving a low-melting glassy
solid. No 13C NMR data were reported at that time. Even then [23], a small amount
of 3c was observed but was not isolated or characterized. Also, in the previous
0
article [23], the minor isomer isolated was identified as aN,xN,x N-tris(tert-
butoxycarbonyl)-L-arginine. Few 1H NMR data were reported, but the d value of the
signal corresponding to the dCH2 group (3.95 ppm) is peculiar for dCHd2NBoc) [23]
0
of 3b and not for dCH2NH (d \ 3.5 ppm [15, 16, 26]) of aN,xN,x N-tris(tert-
butoxycarbonyl)-L-arginine, which provides evidence against such a structure.
On the other hand, there are reports0 [27, 28] that suggest significant instability in
a
a
a basic environment for both N,xN,x N-tris-Cbz-protected and N,dN,xN-tris-Cbz-
protected arginine derivatives. They would tend to lose one protecting group,
a
resulting in N,xN-bis-Cbz-protected derivatives, analogous to 3c, thus explaining
0
its formation, the total absence of the desired aN,xN,x N-tris(tert-butoxycarbonyl)-L-
arginine and the very low yield of 3a. As expected, any further attempt to transform
3c into the tris-tert-butoxycarbonyl arginine derivative by the addition of Boc2O in
large excess under basic conditions yielded no positive results, even at prolonged
reaction times. Finally, the presence of small amounts of the regioisomer 3b can
conceivably be justified by its tendency to convert into the more stable tautomer 3a,
as confirmed by the calculated energy values (Fig. S27 in Online Resource) for the
more stable structure of 3a (- 28.4117 kcal/mol) and the less stable structure of 3b
(- 8.1970 kcal/mol). In a second attempt, we started from the commercially
a
available N-tert-butoxycarbonyl-L-ornithine (4) or from a sample of 4 that we
a
prepared by catalytic hydrogenation of N-tert-butoxycarbonyl-dN-Cbz-L-ornithine
[20, 21] as suggested by another reported procedure [15]. Compound 4 was then
reacted with commercially available 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-
thiopseudourea (5) in a mixture NaHCO3/H2O/dioxane (Scheme 2).
This method was similar to the second protocol described in the literature [12],
but employed the much more expensive bis(tert-butoxycarbonyl)-guanidine
derivative and promised much lower yields. In this case, the correct regioisomer
0
aN,xN,x N-tris(tert-butoxycarbonyl)-L-arginine was obtained but as a mixture of two
rotamers E/Z (2.2/1 as estimated by 1H NMR analysis) in the form of a white solid
that we designated 6 E, Z, from which we managed to separate 6 E from 6 Z as a
white solid by PLC performed on 0.5-mm, 20 9 20 cm plates (CHCl3/MeOH 9/1).
The assignment of E geometry to the more stable, major rotamer was made on
the basis of the calculated energy ( - 18.6265 kcal/mol) for this conformation,
which was lower than that calculated for Z (- 13.9327 kcal/mol) (Fig. S28 in
1
Online Resource). The H NMR spectrum of the mixture showed split peaks for
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1821
SCH3O
N
O
NaHCO3/H2O
dioxane
HN
O
HO
NH2
+
48 h/40 °C
HN
O
O
O
5
4
O
O
O
O
N
N
O
O
N
O
HO
NH
+
HO
N
H
NH
H
HN
O
O
O
HN
O
O
O
O
O
Z (cis) (12%)
E (trans) (27%)
6 (39%)
0
Scheme 2 Synthetic procedure suggested [15] for the preparation of aN,xN,x N-tris(tert-
butoxycarbonyl)-L-arginine and the obtained products
several signals. In particular, for the CH3 resonances derived from Boc groups, there
were three more intense signals at 1.44, 1.48 and 1.49 ppm, which agreed perfectly
with the signals of the single rotamer commonly described in the literature [12–16]
and the signals of isolated 6 E, as well as other less significant signals at 1.43, 1.51
and 1.52 ppm that belong to the less stable rotamer and were later also detected in
the spectrum of the isolated 6 Z that had not been reported previously. As expected,
their total integral value corresponds to 27 protons. In addition, in the proton
a
spectrum, two doublets for the NH and two multiplets for theaCH were visible at
5.4 and 5.7 ppm and at 4.2 and 4.3 ppm, respectively, while in the 13C NMR
spectrum, double signals for the CH3 of the Boc groups were detected (Fig. 3).
1H NMR spectra recorded over time were identical, thus implying that at room
temperature, the mixture was stable, and that the rotamers did not interconvert over
time. However, because we wanted to more thoroughly investigate the potential
interconversion of 6 E/Z, we kept one sample that was dissolved in chloroform and
another in the solid phase for 6 months at room temperature, at which point the solid
sample was dissolved in DMSO-d6, and new 1H and 13C NMR spectra were
Fig. 3 Selected portions of the 1H NMR and 13C NMR spectra of the mixture 6 E, Z
123

1822
S. Alfei, S. Castellaro
acquired. Next, both samples were heated in solution at 100 °C for 48 h and
subsequently cooled to room temperature and examined by NMR. Finally, we
heated the samples at 100 °C for an additional 48 h and recorded their NMR spectra
at 70 °C. The results of this investigation are shown in Fig. 4.
In Fig. 4a, it is possible to observe how the 1H and 13C NMR spectra of a solution
of the E/Z mixture in CDCl3 stored for 6 months at room temperature appear
identical to those acquired earlier (see also Fig. 3), and as the spectra of a solid
sample stored for the same amount of time and then dissolved in another solvent
show no significant divergence, with the exception of some differences in the
chemical shifts, observed primarily in the carbon spectrum. In Fig. 4b it can be
observed that the composition of the mixture remains the same even after heating at
100 °C in both solvents, if cooled to room temperature before the new acquisition,
showing only enhanced resolution with regard to the proton spectrum. Significant
changes are observed for both samples if the spectra are recorded at 70 °C (Fig. 4c).
The less stable Z form is now absent, having been converted to the more
stable E form. It can be rationally concluded that the mixture obtained from the
Fig. 4 Selected portions of the 1H NMR and 13C NMR spectra of the 6 E, Z mixture in CDCl3 and
DMSO-d6 acquired at room temperature after storage for 6 months at room temperature (a), after heating
in solution for 48 h at 100 °C (b), and acquired at 70 °C after heating in solution for 48 h at 100 °C (c)
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1823
reaction is stable both at room temperature and after prolonged heating, as the less
stable Z form converts to the E isomer, which is ultimately the only product
observed. Furthermore, this process is reversible, and after cooling, the mixture
reverts to a composition similar to that of the starting material.
Thus, the results obtained and the calculated energy values associated with the
structure of the five isoforms (see Online Resource, pp. 19, 20, Figs. S27 and S28)
provide evidence of the stability of the three isoforms that were not tested
experimentally under the same conditions. It is logical to assume that compounds 10
and 3a, which are more stable than 6 E and 6Z, will be stable under the same
conditions in the two solvents, while 3b, which is less stable than 6 E and 6 Z, will
be very unstable under the same conditions, thus confirming our established
contention.
To confirm the structures of 6 E and 6 Z, which lack a Boc group bound to thedN
nitrogen atom, in the1H and 13C NMR spectra, the signals corresponding to the
dCHd2NH group were observed at 3.41 ppm (multiplet) and 40.48 ppm, respectively,
as reported previously [15, 16, 26]. A plausible mechanism for the formation of the
E/Z mixture is shown in the Online Resource (Fig. S4).
This procedure was further affected by several complications not reported in the
previous article [15], which contributed to lower yield.
a
•
•
•
•
The crude product contained considerable amounts of unreacted material. N-
tert-butoxycarbonyl-L-ornithine (4) was easily removed by acid washes (0.3 N
HCl), but the thiopseudourea derivative 5 was not easy to remove by simple
crystallization of 6 E, Z.
To obtain 6 E, Z with an acceptable degree of purity and yield (39%), we first
had to crystallize it, which afforded only a small amount of suitable material.
The contaminated residue was then purified via silica gel column chromatog-
raphy using CHCl3/MeOH 9/1 as eluent to remove 5.
The purification by column, however, led to the formation of small amounts of
the anhydride of 6, as assessed by the IR spectra of the material eluted, which
displayed a minor peak corresponding to the presence of the O=C–O–C=O
group at 1794 cm-1
.
Attempts to recover unreacted 4 from acid washing by evaporation of water
under reduced pressure provided a mixture of 4 and its lactam 4a (Fig. 5) in a
roughly 1:1 ratio. The formation of 4a most likely resulted from prolonged
exposure to the high heat necessary for evaporation of the aqueous phase, as
Fig. 5 Structure of lactam 4a
123

1824
S. Alfei, S. Castellaro
a
previously reported [29]. On the other hand, in the procedure for preparing N-
tert-butoxycarbonyl-L-ornithine [20, 21] there was an explicit suggestion to not
exceed 55 °C at reduced pressure during the course of isolating 4.
The proton spectrum of this mixture contained signals characteristic of both 4 and
4a, which was in good agreement with the data reported in the literature for 4a
[30, 31] and 4 [21] at 1.38 ppm (CH3Boc), 1.40–1.80 ppm (bCH2cCH2), 3.80 ppm
a
(aCHNH) and 6.80 ppm (aNH). But peculiar signals associated with the N-tert-
butoxycarbonyl-L-ornithine linear system were also present at 8.37 ppm (dNH2) and
at 2.73 ppm (dCH2NH2) and peculiar signals attributed to the cyclic structure of 4a
were detected at 3.08 ppm (dCH2NH) and at 7.50 ppm (NHC=O). A 1:1 ratio of 4 to
4a was obtained by comparing the integrals of the signals at 2.75 and 3.08 ppm
(Fig. S5 in Online Resource). The signals corresponding to the carbons of dCH2NH2
d
and CH2NH groups of 4 and 4a, which are not detectable in a common 13C NMR
because they are hidden by the solvent (DMSO-d6), became visible up down at
39.30 ppm and 41.20 ppm in a DEPT-135 experiment. Four signals at 156.28,
156.30, 158.21 and 175.19 ppm for four different C=O groups were detected in
the13C NMR spectrum, corresponding to two carbamate C=O, one lactam C=O and
0
a
one acid C=O (Fig. S5 in Online Resource). The goal of preparing N,xN,x N-
tris(tert-butoxycarbonyl)-L-arginine as a single compound and in high yield was
achieved only by applying another reported procedure [16], shown in Scheme 3.
A sample of 4 that we had prepared was added to 1,3-bis(tert-butoxycarbonyl)-2-
(trifluoromethylsulfonyl)guanidine (9) (BBGT) in the presence of N,N-diisopropy-
lethylamine (DIEA) in MeOH [16], and the resulting mixture was stirred for 7 days
0
at room temperature to afford aN,xN,x N-tris(tert-butoxycarbonyl)-L-arginine in
quantitative yield (99%). However, in contrast to what was previously reported [16],
this material was structured as the symmetrical compound 10 (tautomer of 6), based
on NMR spectral data and as the symmetrical structure of 9 had suggested. The
product was obtained as a white crystalline solid and not as a colorless oil [16]. The
BBGT (9) was prepared starting from commercial guanidine hydrochloride (7),
which was added to Boc2O/H2O/dioxane/NaOH, providing the symmetrical
intermediate 1,3-bis(tert-butoxycarbonyl)guanidine (8) (BBG) in 60% yield. This
material was reacted with trifluoromethanesulfonic anhydride/Et3N/DCM, provid-
ing the desired product 9 in 70% yield (Scheme 4) [22]. The structures of
intermediates 8 and of 9 were confirmed by NMR spectra, including DEPT-135,
O
O
N
CF3
S
O
O
O
HN
O
O
O
DIEA/MeOH
7gg/r.t.
+
HO
NH2
HO
O
N
NH
O
N
H
NH
HN
O
NH
O
O
O
O
O
4
10 (99%)
9
0
Scheme 3 Synthetic procedure for preparing aN,xN,x N-tris(tert-butoxycarbonyl)-L-arginine [16] and the
obtained product 10
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1825
O
CF3
S
NaOH/dioxane
(F3CSO2)2O
Et3N/CH2Cl2
O
NH O
O
N
NH
O
H2O/Boc2O
HCl
NH2
O
N
H
N
O
O
N
H
NH
-78 °C r.t.
4h/r.t.
H2N
24h/r.t.
H
7
O
O
8 (60%)
9 (70%)
1,3-bis(tert-butoxycarbonyl)-2-
Scheme 4 Synthetic
procedure
for
(trifluoromethylsulfonyl)guanidine (9) (BBGT) [22]
preparing
and by elemental analysis, and the NMR data were in absolute agreement with the
literature.
For the preparation of 10, shorter reaction times were required if non-polar
solvents such as DCM were used [22]. However, to avoid the conversion of 4 in the
methyl(trimethylsilyl) derivative, and given that 4 is not soluble in DCM, we
elected to use the longer experiment employing MeOH as solvent [16].
0
The NMR spectra of the obtained aN,xN,x N-tris(tert-butoxycarbonyl)-L-arginine
did not show split peaks, confirming that the compound was obtained as a single
isomer, and only the symmetrical structure of 10 which did not involve geometric
isomers was consistent with the spectrum. A plausible mechanism for the formation
of 10 is provided in the Online Resource (Fig. S6).
The unreported structure of 10 was confirmed by other features of the NMR
d
spectra. The presence of CH2–dN=C double bond was confirmed by the shape of
d
the CH2–dN=C signal at 3.42 ppm in the proton spectrum. It was a broad triplet
c
because it was coupled only with the two CH2 protons and not the NH proton.
Otherwise, in the case of the cCH2 CH2–dNH–C = group of 6, the signal appeared to
d
be a broad and shapeless (Fig. S7 in Online Resource). In the 13C NMR spectrum,
d
we identified the carbon signal of the CH2N=C group at 161.94 ppm (161 ppm in
the literature [25] for similar functional groups). Finally, the d values of the signals
of the CH3Boc group in the13C NMR spectrum (28.01, 28.11 and 28.29 ppm) were
different from the d values of both 6 E (28.07, 28.24 and 28.36 ppm) and 6
Z (27.95, 28.24 and 28.44 ppm), confirming the difference in the structure of 10.
The energy calculated for the structure of 10 (- 25.641 kcal/mol), which was lower
than the energies calculated for
6
E
(- 18.6265 kcal/mol) and
6
Z (- 13.9327 kcal/mol), confirmed that 10 was the more stable tautomer, thus
explaining the obtainment of only this compound in quantitative yield (Fig. S28 in
Online Resource).
All the obtained compounds were soluble in all common organic solvents except
for n-pentane, n-hexane, petroleum ether, cyclopentane and cyclohexane, and they
showed poor solubility in diisopropyl ether (DIPE) and were not soluble in water.
1
For the reader’s convenience, the tables below report all the H NMR and 13C
NMR chemical shifts for compounds 3a, 3b, 3c, 6 E, 6 Z and 10 (Tables 1 and 2),
along with the most relevant differences between the1H NMR and 13C NMR
chemical shifts for the same compounds using 10 as reference compound (Tables 3
and 4).
123

1826
S. Alfei, S. Castellaro
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1827
123

1828
S. Alfei, S. Castellaro
Table 3 The most relevant differences in 1H NMR chemical shifts [d (ppm)] of compounds 3a, 3b*, 3c,
6 E, 6 Z and 10
Compound aNH (ppm)#
aCHNH d (ppm)#
(Dppm)§
dCH2 d (ppm)#
(Dppm)§
CH3 Boc d (ppm)#
(Dppm)§
(Dppm)§
10
5.39
? 0.47
? 0.32
4.26
? 0.04
- 0.10
3.42
? 0.45
? 0.45
1.44, 1.48, 1.50
3a
0, ? 0.01, ? 0.02
3b*
- 0.01, ? 0.01,
? 0.01
3c
? 0.21
- 0.02
? 0.31
- 0.16
? 0.03
- 0.06
? 0.07
- 0.01
- 0.01
- 0.01, –, ? 0.02
0, 0, - 0.01
6 E
6 Z
- 0.01, ? 0.03,
? 0.02
*Derived from the NMR spectra of the mixture 3a ? 3b by subtracting the peaks belonging to the
isolated compound 3a
Used as reference compound
#Referred to reference compound
§Referred to all other compounds
Discussion
In addition to the double bond (C=NH) of the guanidine group, L-arginine has four
nitrogen atoms which can more or less avidly bind the protector Boc group,
meaning that the N,N,N-tris(tert-butoxycarbonyl)-L-arginine may exist as more than
one isomer, including regioisomers, rotamers and tautomers. The nitrogen in the a
position is the most reactive; thus, when L-arginine was added to Boc2O under basic
conditions, aN-tert-butoxycarbonyl)-L-arginine (2) was formed in good yield.
0
a
However, when we attempted to convert 2 into the widely documented N,xN,x N-
tris(tert-butoxycarbonyl)-L-arginine that we desired as a single product, in contrast
to literature procedures [12–16], which promised this goal, we observed an
unexpected complex mixture of products involving the formation of five isomers,
with each type of structure dependent on the procedure used, and which we have
designated 3a, 3b, 6 E, 6 Z and 10. Using the procedure that entails employing
Boc2O under basic conditions starting from 2 [12], the desired reported isomer
aN,xN,x N-tris(tert-butoxycarbonyl)-L-arginine was never obtained. In its place a
0
mixture of two regioisomers, 3a and 3b, was achieved. This result can be explained
0
by considering the basicity of the nitrogen atoms (x N [ dN [ xN). The most basic
nitrogen in the x’ position was the first to react with Boc2O, followed by the
nitrogen in the d position, leading to the formation of 3b, which however tended to
provide the more stable tautomer 3a, with the compound bearing a Boc group in the
x’ position remaining in trace amounts. The low stability of 3b was confirmed by
experiments employing the 6 E/Z mixture as reference. The reported lability of Boc
groups in the x’ or d position under basic conditions [27, 28] instead explains the
0
a
total absence of N,xN,x N-tris(tert-butoxycarbonyl)-L-arginine, the presence of 3c
and the very low yield of 3a. However, 3a, once isolated, is stable over time and
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1829
123

1830
S. Alfei, S. Castellaro
with heating in CDCl3 and in DMSO-d6 on the basis of the stability experiments
using the mixture 6 E/Z as reference and considering the calculated energy value
associated with its structure. Starting from aN-tert-butoxycarbonyl-L-ornithine, only
the use of non-basic conditions and reagents bearing two Boc groups (5 or 9)
a
enabled the synthesis of N,xN,x’N-tris(tert-butoxycarbonyl)-L-arginine. However,
0
using the commercial reagent 5 [15], aN,xN,x N-tris(tert-butoxycarbonyl)-L-arginine
was not obtained as a single isomer, but was instead formed as a stable mixture of E/
Z rotamers (6 E, Z), which did not interconvert over time at room temperature or
after prolonged heating in different solvents, as confirmed by NMR investigations.
Isolation of the major rotamer 6 E and the unreported 6 Z was achieved only after
careful chromato0graphic separation by PLC. The third procedure tested [16] gave
rise to aN,xN,x N-tris(tert-butoxycarbonyl)-L-arginine as a single product, as
desired, and in high yield. However, the unreported symmetrical structure of 10
was observed without the involvement of geometric isomers, and this structure was
stable over time and upon heating in both CDCl3 and DMSO-d6 on the basis of the
stability experiments using the mixture 6 E/Z as reference and considering the
calculated energy value associated with its structure. Preliminary experimental
results of a work currently in progress validate these discoveries, showing that the
potential reactivity of the arginine derivatives depends on their structure.
Conclusions
In contrast to what w0as reported in the literature [12–16], which assured the
a
preparation of N,xN,x N-tris(tert-butoxycarbonyl)-L-arginine as a single product,
five isomers were isolated. Specifically, 3a ? 3b, formed as a 2.5:1 mixture (19%),
and 3a, in isomerically pure form (5%), were found to be stable over time and upon
0
heating. The compound aN,xN,x N-tris(tert-butoxycarbonyl)-L-arginine was not
obtained when following the first of the selected literature protocols. A mixture of 6
E, Z (39%) that was stable over time and upon heating, the already reported 6
E (28%) and the unknown 6 Z (10%) were isolated via the second protocol. The
unknown compound 10 (tautomer of 6), which was found to be stable over time and
upon heating, was formed in 99% yield using the third set of experimental
conditions employing the symmetrical reagent (9). Careful chromatography allowed
separation of 3a ? 3b from 3c, and also enabled purification of the 6 E, Z mixture.
The major compound 3a was isolated from the mixture 3a ? 3b, and separation and
isolation of 6 E and 6 Z was achieved. A detailed NMR analysis was conducted with
particular attention devoted to the conformational aspects of the guanidine double
bond (C=N) and its positions, and an extensive survey of the literature confirmed the
interpretation of the collected data and enabled a complete characterization of all
compounds. The reproducibility of the outcomes was substantiated by repeating the
experiments several times. A preliminary research program investigating the
reactivity of all the isomers obtained from esterification reactions is currently in
progress, and initial results have shown that their reactivity is influenced by their
structure. Some unreported complications related to the procedures followed have
also been highlighted.
123

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms…
1831
Acknowledgements This work was financially supported by University of Genoa (Progetti di Ricerca di
Ateneo). The authors wish to thank Dr Gaby Brice Taptue for language assistance and Mr Gagliardo
Osvaldo for elemental analysis. A special thanks to Professor Andrea Spallarossa for calculating the
energies of the tautomers and rotamers, contributing to the realization of Figs. S27 and S28 in the Online
Resource, and providing professional advice.
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